Frequency of Frequency of Tetracycline Resistance Genes Tetracycline Resistance Genes

in Bacterial Genomic DNA in Bacterial Genomic DNA of Swine Fecesof Swine Feces

Sharise Redmond & Jeannette NguyenSharise Redmond & Jeannette Nguyen

Under the Direction of Candace GlendeningUnder the Direction of Candace Glendening

AntibioticsAntibiotics ABXABX

• Agents made by bacteria/mold to inhibit bacterial Agents made by bacteria/mold to inhibit bacterial growthgrowth

Do NOT kill virusesDo NOT kill viruses

• UseUse treat infections in humans and animalstreat infections in humans and animals growth promotion in animalsgrowth promotion in animals

BacteriaBacteria • PathogenicPathogenic - harmful/disease causing - harmful/disease causing

e.g. Salmonella, Escheria coli e.g. Salmonella, Escheria coli O157:H7O157:H7• BeneficialBeneficial – non harmful – non harmful

Commensal - symbiotic relationship which benefits one species Commensal - symbiotic relationship which benefits one species while the other is unaffectedwhile the other is unaffected

lots of commensal bacteria in gut (lots of commensal bacteria in gut (E. coli spp.)E. coli spp.)

History of AntibioticsHistory of Antibiotics 1800’s:1800’s: “germ theory” “germ theory”

• Began search for ABXBegan search for ABX 1929:1929: Fleming discovered PenicillinFleming discovered Penicillin

• 1942 11942 1stst large scale use of Penicillin large scale use of Penicillin• Used largely during WWIIUsed largely during WWII

1946:1946: Penicillin widely available clinically Penicillin widely available clinically• Obtainable OTC by the public until the mid ‘50sObtainable OTC by the public until the mid ‘50s

Dev’t of more ABX over next few decadesDev’t of more ABX over next few decades 1970’s:1970’s: Antibiotic Resistance (AR) recognized Antibiotic Resistance (AR) recognized

as a real threat as a real threat • Meningitis & gonorrhea strains resistant to penicillinMeningitis & gonorrhea strains resistant to penicillin

AB ResistanceAB Resistance Bacteria’s ability to produce a protein that:Bacteria’s ability to produce a protein that:

• disables an ABX or disables an ABX or • prevents transport of the ABX into the cellprevents transport of the ABX into the cell

Main hypothesis of AR:Main hypothesis of AR:

penicillinpenicillin flouroquinolonesflouroquinolonesSOONSOON

cephalosporinscephalosporinstetracyclinetetracycline

Genetic Genetic mutationsmutations

N. gonorrhoeaeN. gonorrhoeae (gonorrhea) (gonorrhea) resistance:resistance:

+ antibiotics in + antibiotics in environmentenvironment

= antibiotic = antibiotic resistanceresistance

ABX Use in AnimalsABX Use in Animals

Therapeutic – treatment of bacterial Therapeutic – treatment of bacterial infectioninfection

Sub-therapeutic levels – prevention of Sub-therapeutic levels – prevention of disease & growth promotiondisease & growth promotion

Antibiotics used for growth promotion Antibiotics used for growth promotion pigs gain weight:pigs gain weight:

3.3-8.8% increased weight3.3-8.8% increased weight 2.5-7.0% feed efficiency2.5-7.0% feed efficiency

• Food Research Institute, Doyle, 1998Food Research Institute, Doyle, 1998

ABX use by farmers is not regulatedABX use by farmers is not regulated ~25 million pounds annually used ~25 million pounds annually used

Does the use growth Does the use growth promotional levels of promotional levels of ABX in food animals ABX in food animals

lead to AR bacteria in lead to AR bacteria in our food?our food?

History of TetracyclineHistory of Tetracycline 19481948:: introduction of tetracycline introduction of tetracycline

• Made by Made by Streptomyces bacteriumStreptomyces bacterium in soil in soil• Chemical structure:Chemical structure:

• ““broad spectrum” broad spectrum” • low toxicitylow toxicity

1953:1953: Shigella dysenteriae dev. resistance to tetracycline Shigella dysenteriae dev. resistance to tetracycline Today 2Today 2ndnd to penicillin in the world in production and to penicillin in the world in production and

useuse• Treat: Respiratory tract infections, typhus, cholera, Treat: Respiratory tract infections, typhus, cholera,

brucellosis, anthrax, syphilis, Chlamydia, acne brucellosis, anthrax, syphilis, Chlamydia, acne • Also used widely for growth promotion in animals.Also used widely for growth promotion in animals.

Tet Mechanisms of ActionTet Mechanisms of Action Tetracycline inhibits Tetracycline inhibits

bacterial growth by bacterial growth by inhibiting inhibiting translation. translation. • It binds to the It binds to the

ribosomal subunit ribosomal subunit and prevents the and prevents the amino-acyl tRNA amino-acyl tRNA from binding to the A from binding to the A site of the ribosome.site of the ribosome.

Inhibition of Protein Inhibition of Protein Synthesis by TetracyclineSynthesis by Tetracycline

Mechanisms of Tet ResistanceMechanisms of Tet Resistance

Ribosomal Ribosomal Protection Protection ProteinProtein

plasmidplasmid

Efflux PumpEfflux Pump

Inactivation Inactivation EnzymeEnzyme

TetracyclineTetracycline

Effluxn = 23

Ribosomal protectionn = 11

Enzymatic Inactivationn = 3

Unknownn = 1

tet(A), tet(B), tet(C), tet(D),tet(E) tet(G), tet(H), tet(J), tet(V), tet(Y), tet(Z), tet(30), tet(31), tet(K), tet(L), tetA(P), otr(B), tcr3 tet(33), tet(35), tet(38), tet(39), otr(C)

tet(M), tet(O), tet(S), tet(W),tet(Q), tet(T), otr(A), tetB(P)b, tet, tet(32), tet(36

tet(X), tet(34), tet(37)

tet(U)

Mechanism of resistance for characterized tet and otr genes

Growth PromotionGrowth Promotion

There has been a lack of serious There has been a lack of serious studies in the amounts of antibiotics studies in the amounts of antibiotics given to livestock and its link to the given to livestock and its link to the increasing rates of resistance genes.increasing rates of resistance genes.

Previously Done StudiesPreviously Done StudiesOrganism Organism of Originof Origin

Organism Organism StudiedStudied

# Tet Res # Tet Res GenesGenes

# # SamplesSamples

ReferenceReference

Human & Human & various various animalsanimals

E. coliE. coli tettet: A, B, C, D, : A, B, C, D, E, G, K, L, M, O, E, G, K, L, M, O, S, A(P), Q, XS, A(P), Q, X

12001200 Bryan et al. Bryan et al. (2004)(2004)

SwineSwine LactobacillusLactobacillus tettet: M: M 9494 Gevers et al. Gevers et al. (2002)(2002)

SwineSwine E. coliE. coli tet: A, B, C, D,E, G, H, J, Y, Z, 30

2121 Aminov et al. Aminov et al. (2002)(2002)

BovineBovine A. pyogenesA. pyogenes tettet: W: W 2020 Billington et Billington et al. (2002)al. (2002)

HumanHuman oral microfloraoral microflora tettet: M, W, O, Q, : M, W, O, Q, S, L, A, KS, L, A, K

105105 Villedieu et al. Villedieu et al. (2002)(2002)

Groundwater Groundwater outflow from outflow from swine farmswine farm

Soil & Soil & gastrointestinal gastrointestinal microbiotamicrobiota

tettet: O, Q, W, M, : O, Q, W, M, P, S, T, P, S, T, otrotrAA

2222 Chee-Sanford Chee-Sanford

et al. (2001)et al. (2001)

Central QuestionCentral Question

Does the use of tetracycline as a Does the use of tetracycline as a growth promotant affect tetracycline growth promotant affect tetracycline resistance in swine fecal flora?resistance in swine fecal flora?

Central HypothesisCentral Hypothesis

The use of tetracycline as a growth The use of tetracycline as a growth promotant will promotant will frequency of detecting frequency of detecting Tet Resistance Genes in swine fecal flora.Tet Resistance Genes in swine fecal flora.

Effects of Growth Promotional use Effects of Growth Promotional use of Chlorotetracycline (CTC)of Chlorotetracycline (CTC)

Large-scale, multi-year study led by Large-scale, multi-year study led by Julie Funk @ Ohio State Univ. (OSU)Julie Funk @ Ohio State Univ. (OSU)

Epidemiological approach to studying Epidemiological approach to studying the use of CTC as a growth the use of CTC as a growth promotant for swinepromotant for swine

Looked for AR bacteriaLooked for AR bacteria

Treatment from 10 weeks Treatment from 10 weeks ((50 lbs)50 lbs) until 6 months old until 6 months old ((250 lbs)250 lbs)..• Pigs sampled pre-slaughter

• 14 barn pairs total• 96 pigs per barn

• 2688 total pigs sampled

Treatment from 10 weeks Treatment from 10 weeks ((50 lbs)50 lbs) until 6 months old until 6 months old ((250 lbs)250 lbs)..• Pigs sampled pre-slaughter

• 14 barn pairs total• 96 pigs per barn

• 2688 total pigs sampled

CDC Year 1 Study DesignCDC Year 1 Study Design

ControlControl(no antibiotics in the feed)

TreatmentTreatment(50g CTC/ton of feed)

Temporally matched Barn PairTemporally matched Barn Pair

Selected CDC Year 1 ResultsSelected CDC Year 1 Results

Isolated 100 Isolated 100 different Gram different Gram Negative bacteria Negative bacteria (usually (usually E. coliE. coli) ) from each fecal from each fecal samplesample

Studied resistance Studied resistance to 4 antibioticsto 4 antibiotics

Found phenotypic Found phenotypic (tet res) difference (tet res) difference between these 2 between these 2 groupsgroups

1.01.0

.90.90

.80.80

.70.70

.60.60

.50.50

.40.40

.30.30

.20.20

.10.10

00P

rop

ort

ion

Res

ista

nt

to C

TC

Pro

po

rtio

n R

esis

tan

t to

CT

CTreatmentTreatment

No CTCNo CTC CTCCTC

Gram Negative Gram Negative Fecal Flora IsolatesFecal Flora Isolates

n=268,800 isolates

ObjectivesObjectives To study the distribution of tetracycline resistance To study the distribution of tetracycline resistance

genes found in the fecal flora of pigsgenes found in the fecal flora of pigs• + CTC diet in their finishing phase.+ CTC diet in their finishing phase.• Ctrl: NO growth promotional use of ABXCtrl: NO growth promotional use of ABX

Our Study PopulationOur Study Population• 10 barn pairs• 48 pigs per barn

480 total pigs sampled Recall there are at least 38 tet resistance genesRecall there are at least 38 tet resistance genes

• Are certain genes found more often under the selective Are certain genes found more often under the selective pressure of tetracycline?pressure of tetracycline?

ExperimentalExperimentalDesignDesign

200 200 l genomic DNA l genomic DNA (from bacterial population)(from bacterial population)

Qiagen Stool DNA Extraction Kit

(Bacterial Genomic DNA)

Multiplex PCRMultiplex PCR

Group 3Group 3 Group 2Group 2 Group 4Group 4Group 1Group 1

1 1 ll 1 1 ll 1 1 ll 1 1 ll

200 mg poop 200 mg poop (frozen quickly)(frozen quickly)

Methods: Multiplex PCRMethods: Multiplex PCR 2 (or more) sets of 2 (or more) sets of

primers in same tubeprimers in same tube• Ex: Group 1: Ex: Group 1:

tettet(B) 659 bp(B) 659 bp tettet(C) 418 bp(C) 418 bp tettet(D) 787 bp(D) 787 bp

Run each sample through Run each sample through four separate Multiplex four separate Multiplex PCR reactions. PCR reactions. 480 samples x 4 480 samples x 4

groups = 1920 rxns!groups = 1920 rxns!

E-gel Marker

2000800

400

200

100

B/C/DB C D

Ng et al., 2001

Genes StudiedGenes StudiedGroup (+) Plasmid Tetracycline

resistance geneAmplicon size

(bp)

1Efflux Pump

pRT11 tet(B) 659

pBR322 tet(C) 418

pSL106 tet(D) 787

2Efflux Pump

pSL18 tet(A) 210

pSL1504 tet(E) 278

pJA8122 tet(G) 468

3Ribosomal Protection OrEfflux Pump

pAT102 tet(K)a 169

pVB.A15 tet(L) 267

pJ13 tet(M) 406

pUOA1 tet(O) 515

pAT451 tet(S) 667

4Ribo. Protection Or Enzyme Inactivation

pJIR39 tetA(P) 676

pNFD13-2 tet(Q) 904

pBS5 tet(X) 468

Sample Gel (Group 3)Sample Gel (Group 3)

667515406267169

(+) Controls _

K L M O S (-)

Individual Pigs from Farm Bailey 3 _

Individual Pigs from Farm Bailey 3 _

Sample Gel (Group 4)Sample Gel (Group 4)

(+) Controls

? Q X (-)

Individual Pigs from Farm Bailey 3 _ _

___ _ Individual Pigs from Farm Bailey 3 _______

904468

The Effect of CTC in Swine Finisher Feed on Detection of Tet Resistance Genes

0

20

40

60

80

100

120

140

160

180

200

CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl

B C D K L M O S

Tet Resistance Gene

# F

ecal S

am

ple

s

Neg

Pos

ResultsResults

ResultsResults

The Effect of CTC in Swine Finisher Feed on Detection of Tet Resistance Genes

0

20

40

60

80

100

120

140

160

CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl

A E G A(P) Q X

Tet Resistance Gene

# F

ecal S

am

ple

s

Neg

Pos

ResultsResults

The Effect of CTC in Swine Finisher Feed on Proportion of Pigs with Tet Resistance Genes

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

B C D K L M O S

Tet Resistance Gene

% P

osit

ive

CTC

no CTC

* *

.33.3 .49 5 E-10 1 E-4

The Effect of CTC in Swine Finisher Feed on Proportion of Pigs with Tet Resistance Genes

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

A E G A(P) Q X

Tet Resistance Gene

% P

osit

ive

CTC

no CTC

* *

*

ResultsResults

6 E-4 2 E-5 3 E-7

Tet Resistance Genes Grouped by Mechanism

0

50

100

150

200

250

300

350

Ribosomal Protection Efflux Enzymatic Inactivation

Tet Resistance Gene Type

# D

ete

cte

d

CTC

ctrl

* *

*

ResultsResults

.008 .03 3 E-7

Central HypothesisCentral Hypothesis

The Effect of CTC in Swine Finisher Feed on # of Pigs with a Detectible Tet Resistance Gene

0

20

40

60

80

100

120

140

160

180

CTC ctrl

Treatment

# P

igs

Pos

Neg

.94

81%81%

The use of tetracycline as a growth The use of tetracycline as a growth promotant will promotant will frequency of detecting frequency of detecting Tet Resistance Genes in swine fecal flora.Tet Resistance Genes in swine fecal flora.

DiscussionDiscussion At least one tet res gene in 81% of both At least one tet res gene in 81% of both

treatment groups treatment groups 5/8 tet res genes showed no statistical diff. btw 5/8 tet res genes showed no statistical diff. btw

treatment groups or were not high in frequencytreatment groups or were not high in frequency• tettet(C), (L), (M)(C), (L), (M) similar high frequency in both swine similar high frequency in both swine

groupsgroups• tettet(B), (D), (K)(B), (D), (K) min. to 0 frequency in both swine min. to 0 frequency in both swine

groupsgroups• tettet(S) mostly found in ctrl samples(S) mostly found in ctrl samples• tettet(O) mostly found in CTC samples(O) mostly found in CTC samples

Group 4 AR genes in both treatment groupsGroup 4 AR genes in both treatment groups• 3/3 tet res genes statistically diff. btw treatment groups3/3 tet res genes statistically diff. btw treatment groups

Most tet genes found in CTC groupsMost tet genes found in CTC groups Group 2 data in progressGroup 2 data in progress

Future WorkFuture Work

Complete sample processingComplete sample processing• 4 more barn pairs4 more barn pairs

Look at more tet resistance genesLook at more tet resistance genes Try to quantitate the amount of each Try to quantitate the amount of each

tet gene present in the sampletet gene present in the sample

ReferencesReferences1.1. Aminov, R. I.; Chee-Sanford, J. C.; Garrigues, N.; Teferedegne, B.; Krapac, I. J.;. White, B. A.; Mackie, R. I. (2002) Aminov, R. I.; Chee-Sanford, J. C.; Garrigues, N.; Teferedegne, B.; Krapac, I. J.;. White, B. A.; Mackie, R. I. (2002)

Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of Gram-Negative Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of Gram-Negative BacteriaBacteria. Applied and Environmental Microbiology, . Applied and Environmental Microbiology, 68(4), 1786-1793. 68(4), 1786-1793.

2.2. Aminov, R. I; Garrigues-Jean, N; Mackie, R. I. (2000) Molecular Ecology of Tetracycline Resistance: Development and Aminov, R. I; Garrigues-Jean, N; Mackie, R. I. (2000) Molecular Ecology of Tetracycline Resistance: Development and Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins. Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins. Applied and Applied and Environmental Microbiology Environmental Microbiology 67 (1), 22-32.67 (1), 22-32.

3.3. Billington, S. J.; Songer, J. G.; Jost, B. H. (2002) Widespread Distribution of a Tet W Determinant among Tetracycline-Billington, S. J.; Songer, J. G.; Jost, B. H. (2002) Widespread Distribution of a Tet W Determinant among Tetracycline-Resistant Isolates of the Animal Pathogen Acranobacterium pyogenes.Resistant Isolates of the Animal Pathogen Acranobacterium pyogenes. Antimicrobial Agents and Chemotherapy, Antimicrobial Agents and Chemotherapy, 1281-1281-1287.1287.

4.4. Bryan, A.; Shapir, N.; Sadowsky, M.J. (2004) Bryan, A.; Shapir, N.; Sadowsky, M.J. (2004) Frequency and Distribution of Tetracycline Resistance Genes in Frequency and Distribution of Tetracycline Resistance Genes in Genetically Diverse, Nonselected, and Nonclinical Genetically Diverse, Nonselected, and Nonclinical Escherichia coliEscherichia coli Strains Isolated from Diverse Human and Animal Sources Strains Isolated from Diverse Human and Animal Sources

5.5. Chee-Sanford, J. C.; Aminov, R. I.; Krapac, I. J.; Garrigues-JeanJean, N.; Mackie, R. I. (2001) Occurrence and Diversity of Chee-Sanford, J. C.; Aminov, R. I.; Krapac, I. J.; Garrigues-JeanJean, N.; Mackie, R. I. (2001) Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities.Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities. Applied and Applied and Environmental MicrobiologyEnvironmental Microbiology, 67(4), 1494-1502., 67(4), 1494-1502.

6.6. Chopra, I.; Roberts, M. (2001) Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Chopra, I.; Roberts, M. (2001) Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial ResistanceBacterial Resistance. Microbiology and Molecular Biology Reviews, . Microbiology and Molecular Biology Reviews, 65(2), 232-260.65(2), 232-260.

7.7. Doyle, M. E. (2001) Doyle, M. E. (2001) Alternatives to Antibiotic Use for Growth Promotion in Animal Husbandry. Food Research Institute: Briefings, University of Wisconsin-Madison 1-17.

8.8. Gevers, D.; Danielsen, M.; Huys, G.; Swings, J. (2002) Molecular Characterization of tet(M) Genes in Lactobacillus Isolates Gevers, D.; Danielsen, M.; Huys, G.; Swings, J. (2002) Molecular Characterization of tet(M) Genes in Lactobacillus Isolates from Different Types of Fermented Dry Sausagefrom Different Types of Fermented Dry Sausage. Applied and Environmental Microbiology, . Applied and Environmental Microbiology, 69(2), 1270-1275.69(2), 1270-1275.

9.9. http://dictionary.reference.comhttp://dictionary.reference.com10.10. http://en.wikipedia.org/wiki/Tetracycline http://en.wikipedia.org/wiki/Tetracycline 11.11. Lefers, Mark and Holmgren Lab (2004) http://www.biochem.northwestern.edu/holmgren/Glossary/Definitions/Def-A/Lefers, Mark and Holmgren Lab (2004) http://www.biochem.northwestern.edu/holmgren/Glossary/Definitions/Def-A/

antibiotic_resistance.htmlantibiotic_resistance.html12.12. Levy, M.D., Stuart B. (2002). Levy, M.D., Stuart B. (2002). The Antibiotic Paradox. The Antibiotic Paradox. Cambridge, MA: Perseus PublishingCambridge, MA: Perseus Publishing13.13. Mathews, K. H. (2001) Antibiotic Drug Use and Veterinary Costs in U.S. Livestock Production. Mathews, K. H. (2001) Antibiotic Drug Use and Veterinary Costs in U.S. Livestock Production. United States Department of United States Department of

Agriculture Economic Research Service, Agriculture Economic Research Service, Agriculture Information Bulletin 766. Agriculture Information Bulletin 766. 14.14. Ng, L.-K.; Martin, I.; Alfa, M.; Mulvey, M. (2001) Multiplex PCR for the detection of tetracycline resistant genes.Ng, L.-K.; Martin, I.; Alfa, M.; Mulvey, M. (2001) Multiplex PCR for the detection of tetracycline resistant genes. Molecular Molecular

and Cellular Probes, and Cellular Probes, 15, 209-215.15, 209-215.15.15. Rubkin, Roberts, Institute of Medicine (1998) Rubkin, Roberts, Institute of Medicine (1998) Antimicrobial Resistance: Issues and Options. Antimicrobial Resistance: Issues and Options. Washington, DC: Harrison, P. R. Washington, DC: Harrison, P. R.

and Lederberg, J. National Academy Press. and Lederberg, J. National Academy Press. 16.16. Villedieu, A.; Diaz-Torres, M. L.; Hunt, N.; McNab, R.; Spratt, D. A.; Wilson, M.; Mullany, P. (2002) Prevalence of Tetracycline Villedieu, A.; Diaz-Torres, M. L.; Hunt, N.; McNab, R.; Spratt, D. A.; Wilson, M.; Mullany, P. (2002) Prevalence of Tetracycline

Resistance Genes in Oral Bacteria.Resistance Genes in Oral Bacteria. Antimicrobial Agents and Chemotherapy, Antimicrobial Agents and Chemotherapy, 47(3), 878-882.47(3), 878-882.17.17. White, D.G.; Zhao, S.; Simjee, S.; Wagner, D. D.; McDermott, P. F. (2002)White, D.G.; Zhao, S.; Simjee, S.; Wagner, D. D.; McDermott, P. F. (2002) Antimicrobial resistance of foodborne pathogensAntimicrobial resistance of foodborne pathogens. .

Microbes and Infection Microbes and Infection 4, 405-412.4, 405-412.

Acknowledgements:Acknowledgements: Grant!!!Grant!!! Julie Funk, MS, DVM, PhD, Asst. Prof. @ OSU School Julie Funk, MS, DVM, PhD, Asst. Prof. @ OSU School

of Veterinary Medicineof Veterinary Medicine Fecal Extraction TeamFecal Extraction Team

• Andy BowmanAndy Bowman• Luc HesselschwardtLuc Hesselschwardt• Andy MackAndy Mack• Jodi Houser Jodi Houser • Jamie BerningJamie Berning

Candace GlendeningCandace Glendening Each OtherEach Other University of RedlandsUniversity of Redlands